The formation of dentin by odontoblasts involves the secretion of an uncalcified matirix, predentin consisting of type I collagen and proteoglycans. Before predentin is transformed to dentin and apatite crystals are laid down onto the collagenous matrix, some of the proteoglycans are removed, probably by an enzymatic process. Also some noncollageous proteins (glycoproteins and phosphoproteins) are secreted at the mineralization from and may participate in the transformation of predentin to dentin. The aim of the present studies is to elucidate the nature of the noncollageous proteins and proteoglycans of rat dentin and to study their metabolism and biological activities, in order to understand the roles they might play in dentinogenesis. New methodology has been devised to extract and partially purify the macromolecules without causing artifactual losses of the substances. Several classes of noncollagenous dentin proteins are being studied: highly phosphorylated phosphoproteins, acidic glycoproteins, serum proteins, and gamma-carboxyglutamate-containing proteins. In addition two pools of proteoglycans are being characterized, one extractable prior to demineralization and a second extractable only after decalcification in EDTA. Another major thrust of the research is to develop an odontoblast organ culture system that mimicks in vivo events. The system we are using, consisting of pieces of rat incisors cultured in defined medium, incorporates (3H)-serine into phosphoproteins and other macromolecules and (3H)-proline into (3H)-hydroxyproline in insoluble collagen. There is a steady increase in the radioactivity of these macromolecules in the insoluble matrix for up to five days of culture. We now plan to utilize this culture system as a model for studies on the biosynthesis, secretion and metabolic fate of collagen, noncollagenous proteins and proteoglycans.